Purpose:
The transforming growth factor β (TGF- β) and the Wnt, Notch signalling pathways all play crucial roles in many biological processes, including embryonic development, cell fate differentiation and cell proliferation. There is evidence for a crosstalk between the Notch and Wnt pathways during retinal development, but it is not known whether co-activation of these pathways may occur in adult human Müller stem cells (hMSCs) or whether these can be modulated by TGF- β.It was therefore the aim of this study to investigate the effect of this cytokine on the expression of molecules involved in these two pathways

Methods:
Human Müller stem cells were cultured on matrigel using DMEM containing foetal calf serum (FCS). hMSCs were cultured in the absence or presence of the γ-secretase inhibitor DAPT plus bFGF to induce their differentiation into RGCs. Exogenous TGF- β1 or TGF- β3 were added to the differentiating or control cells which were cultured for up to 7 days. mRNA and protein extracted from these cells were examined by RT-PCR, western-blot and immuno-staining techniques for the expression of molecules of the Notch and Wnt signalling pathways. The hexosaminidase assay and Ki67 immuno-staining were used to test the effect of TGF- β on cell proliferation.

Results:
Notch inhibition by DAPT caused down-regulation in gene expression of Hes1 and Wnt 2b. Similarly, TGF- β1 alone caused a decrease in gene and protein expression of the Notch downstream target Hes1 and the Wnt signalling ligand Wnt2b, while inducing an increase of the canonical Wnt signalling intracellular component β-catenin. Down-regulation of these genes by DAPT was not modified by addition of TGF- β1 or TGF- β3 to the cultured cells. It was also observed that both TGF- β1 and TGF- β3 had a similar effect on the inhibition of cell growth.

Conclusions:
These results provide evidence that TGF- β directly induces down-regulation of the Notch target Hes1 and the Wnt signalling ligand Wnt2b, as well as upregulation of β-catenin. Furthermore, down-regulation of Notch by DAPT results in down-regulation of Wnt2b, suggesting a crosstalk between these two signalling pathways. However, TGF- β does not have additive effects on the down-regulation of Hes1 and Wnt2b by DAPT. The observations suggest that these two molecules may activate similar intracellular pathways mediating neural differentiation of Müller stem cells.